GE Healthcare
Amersham
ECL Direct Nucleic Acid
Labelling And Detection
Systems
Product Booklet
Codes:
RPN3000
RPN3001
RPN3005
Page finder
1. Legal
4
2. Handling
2.1. Safety warnings and precautions
2.2. Storage
2.3. Stability
5
5
5
5
3. Components of the system
6
4. ECL direct nucleic acid labelling and detection systems
7
5. Description
8
6. Critical Parameters
11
7. Additional equipment and solutions required
7.1. Equipment
7.2. Solutions
12
12
12
8. The ECL direct nucleic acid labelling and detection system
protocols
8.1. Protocol summary
8.2. Protocol A. Labelling, hybridization and detection
8.2.1. Preparation of labelled probe
8.2.2. Hybridization and stringency washes
8.2.3. Signal generation and detection
8.2.4. Hybridization and stringency washing in tubes
8.3. Protocol B. Southern blotting
8.3.1. Gel electrophoresis
8.3.2. Processing the gel
8.3.3. Capillary blotting
8.3.4. Processsing the blot
8.3.5. Probe labelling, hybridization and detection
8.4. Protocol C. Northern blotting
8.4.1. Gel electrophoresis
8.4.2. Treating the gel
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15
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19
23
26
30
30
32
33
35
36
37
37
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2
8.4.3. Blotting the gel
8.4.4. Probe labelling, hybridization and detection
8.5. Protocol D. Colony and plaque blotting
8.5.1. Plating out the incubation
8.5.2. Colony/plaque lifts
8.5.3. Lysis and fixation
8.5.4. Rinsing
8.5.5. Hybridization and detection
9. Additional information
9.1. Quality control
9.2. Rebrobing blots
9.3. Storage of labelled probes
9.4. Excising inserts from vectors
9.5. Labelling probes in low melting point agarose
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42
43
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10. Troubleshooting
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11. References
51
12. Related products
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3
1. Legal
GE and GE Monogram are trademarks of General Electric Company.
Amersham, ECL, Hyperfilm, Hybond and Sensitize are treademarks of
GE Healthcare companies.
SaranWrap is a trademark of the Dow Chemical Co
Whatman is a trademark of Whatman Paper Ltd
Pipetman is a trademark of Gilson
© 2006 General Electric Company – All rights reserved.
GE Healthcare reserves the right, subject to any regulatory and
contractual approval, if required, to make changes in specification
and features shown herein, or discontinue the product described at
any time without notice or obligation.
Contact your GE Healthcare representative for the most current
information and a copy of the terms and conditions.
www.gehealthcare.com/lifesciences
GE Healthcare UK Limited
Amersham Place, Little Chalfont,
Buckinghamshire, HP7 9NA UK
4
2. Handling
2.1. Safety warnings
and precautions
You are reminded that certain
components in the solutions
may cause bleaching on
contact with skin.
Warning: For research use
only. Not recommended
or intended for diagnosis
of disease in humans or
animals. Do not use internally
or externally in humans or
animals.
2.2. Storage
Store hybridization buffer
and blocking agent at room
temperature. Store remainder
of kit at 2–8°C.
All chemicals should be
considered as potentially
hazardous. We therefore
recommend that this product is
handled only by those persons
who have been trained in
laboratory techniques and
that it is used in accordance
with the principles of good
laboratory practice. Wear
suitable protective clothing
such as laboratory overalls,
safety glasses and gloves.
Care should be taken to avoid
contact with skin or eyes. In
the case of contact with skin
or eyes wash immediately
with water. See material safety
data sheet(s) and/or safety
statement(s) for specific advice.
2.3. Stability
This system is stable for
3 months when stored under
the recommended conditions.
5
3. Components of the system
RPN3000
1. DNA labelling reagent
0.5 ml
Store at 2–8°C
2. Glutaraldehyde solution
0.5 ml
Store at 2–8°C
3. Water
1.0 ml
Store at 2–8°C
4. l Hind III control DNA
0.1 ml
(10 μg/ml) Store at 2–8°C
5. Gold hybridization buffer 500 ml
Store at room temperature
(15–25°C) prior to use. Once
this has had blocking agent
added it should be stored in
aliquots at -15°C to -30°C
6. Blocking agent
25 g
Store at room temperature
(15–25°C) prior to use.
7. ECL detection reagent 1
125 ml
Store at 2–8°C
8. ECL detection reagent 2
125 ml
Store at 2–8°C
RPN3001
2 x 0.5 ml
RPN3003
0.5 ml
2 x 0.5 ml
0.5 ml
2 x 1.0 ml
1.0 ml
2 x 0.1 ml
0.1 ml
2 x 500 ml
500 ml
2 x 25 g
25 g
2 x 125 ml
-
2 x 125 ml
-
All components are stable for 3 months when stored under the above
conditions.
6
4. ECL direct nucleic acid labelling and
detection systems
Complete systems
Labelling reagents for 5 μg DNA
Hybridization buffer and detection reagent
for 2000 cm2 membrane
Labelling reagents for 10 μg DNA
Hybridization buffer and detection reagent
for 4000 cm2 membrane
Refill packs
ECL direct nucleic acid labelling system
Labelling reagents for 5 μg DNA
Hybridization buffer for 2000 cm2 membrane
ECL detection reagents
Detection reagents for 2000 cm2 membrane
Detection reagent for 4000 cm2 membrane
ECL gold buffer
Hybridization buffer and blocking agent
for 4000 cm2 membrane
7
RPN3000
RPN3001
RPN3005
RPN3004
RPN2105
RPN3006
5. Description
The ECL™ direct nucleic acid labelling and detection system from
GE Healthcare is based on enhanced chemiluminescence (1). The
principle technique is outlined in figure 1, page 9.
The system involves directly labelling probe DNA or RNA with the
enzyme horseradish peroxidase (2). This is achieved by completely
denaturing the probe so that it is in single-stranded form.
Peroxidase, which has been complexed with a positively charged
polymer, is added and it forms a loose attachment to the nucleic
acid by charge attraction. These ionic interactions will be interrupted
by the presence of counter ions so it is important that the probe is in
a low salt solution. Addition of glutarldehyde causes the formation
of chemical cross-links so that the probe is covalently labelled with
enzyme.
Once labelled, the probe is used in hybridization with target DNA or
RNA immobilized on a membrane. From this stage onwards it is
important to take care that the enzyme activity is not lost and
therefore that the temperature of the hybridization mixture never
exceeds 42°C. A specially optimized hybridization buffer is included
in the system which ensures efficient hybridization and protects
the peroxidase during this step. A novel rate enhancer is included
in the buffer to generate additional sensitivity. The formulation of
the buffer includes 6 M urea which is equivalent to 50% formamide
in reducing the Tm of hybridization. Therefore when controlling
stringency of hybridization the only parameter that may be altered is
salt concentration. Recommendations for a suitable initial stringency
are given in the protocol.
After hybridization the filters are washed to remove unlabelled probe,
again taking care to keep the temperature at or below 42°C. Stringency
may be controlled by altering the urea or SSC concentraion in the
primary wash buffer. If urea is omitted, the temperature of this wash
8
Double-stranded DNA probe
Boil. snap cool
Negatively-charged denatured
probe
DNA labelling reagent –
positively-charged complexes
of peroxidase
Mix
Peroxidase loosely bound to
probe
Add glutaraldehyde
Labelled probe – peroxidase
complexes covalently linked to
DNA
Hybridization
Probe DNA bound to target
Detection reagent 1
Detection reagent 2
0 _2 + H 0
2
Luminol
Oxidized product
Enhancer
Light
Results on film
Figure 1. Principles of the ECL direct nucleic acid labelling and
detection system.
9
can be raised to a maximum of 55°C, provided the wash is performed
for no longer than 2 x 20 minutes. The washed filters can then be
taken directly into the ECL detection step. There are two detection
reagents which should be mixed immediately before use.
Detection reagent 1 decays to hydrogen peroxide, the substrate
for peroxidase. Reduction of hydrogen peroxide by the enzyme is
coupled to the light producing reaction by detection reagent 2. This
contains luminol, which on oxidation produces the light. The light
output is increased and prolonged by the presence of an enhancer
so that it can be detected on a blue-light senstive film. The ECL direct
system has been successfuly used in the Southern (3,4), Northern (5)
and colony/plaque blotting applications (6).
This booklet contains protocols to all the steps involved in the ECL
direct system, from blotting to detection (pages 15–42). It is a simple
and rapid technique. It is important, however, to follow the protocol
carefully to ensure good results. The protocol summary gives an idea
of timescales involved with the system. However, it is recommended
that the full protocols are followed until users are familiar with the
system. A detachable protocol summary card is provided at the back
of this booklet.
10
6. Critical parameters
• Hybridization temperature must not exceed 42°C to avoid loss of
enzyme activity.
• Adequate circulation of the buffer is essential during hybridization
as blots should be allowed to move freely.
• Wear powder-free gloves. Powder from the gloves can inhibit the
ECL reaction and result in blank patches on film.
• Avoid cross contamination of detection reagents 1 and 2.
11
7. Additional equipment and solutions
required
7.1. Equipment
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
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•
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•
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Adjustable pipettes for example Pipetman™
Sterile pipette tips
Gel elephoresis equipment (tanks, power units etc)
Heating equipment for agarose, for example microwave oven
Large containers for gel boiling
Standard laboratory glassware
Filter paper (for example, Whatman-3MM)
Waterbaths, 37°C and 42°C shaking
Ultraviolet transilluminator
Orbital shaker
SaranWrap (or other suitable cling film)
Paper towels
Vacuum oven
Polypropylene microcentrifuge tubes
Vortex mixer
Microcentrifuge
Plastic boxes/bags for hybridization
Forceps with rounded non-serrated tips
Plastic screw-top universal bottles
X-ray film-cassettes
Timer
Film developing facility and reagents
7.2. Solutions
Solutions that are used for DNA electrophoresis, for blotting and
for washing the blots after hybridization that are not supplied with
the ECL direct nucleic acid labelling and detection system are listed
12
here. It is recommended that analytical grade reagents and doubledistilled water are used throughout.
50x TAE buffer for agarose
Tris (base)
2 M (242 g)
EDTA (disodium salt)
0.05 M (18.6 g)
Adjust to pH8 with glacial acetic acid (~57 ml) and make up to 1 litre.
For use, dilute x50 to give Tris acetate 0.04 M and EDTA 0.001 M.
Reagent for loading DNA samples on to gels
Bromophenol blue
0.05 g
Xylene cyanol
0.05 g
Glycerol
5.00 ml
EDTA
0.186 g
Make up to 10 ml with TAE buffer
Loading buffer for RNA samples
Formamide
0.72 ml
10 x MOPS buffer
0.16 ml
Formaldehyde (37%)
0.26 ml
Water
0.18 ml
80% glycerol
0.1 ml
Bromophenol blue
(saturated solution)
0.08 ml
Store in a freezer at -15°C to -30°C.
10x MOPS buffer
MOPS [3-(N-morpholino)
propanesulphonic acid]
Sodium acetate
EDTA
0.2 M
0.05 M
0.01 M
Depurination solution
HCl
250 mM
13
Denaturation solution
NaCl
NaOH
1.5 M
0.5 M
Neutralizing solution
NaCl
Tris HCl
pH adjusted to 7.5
1.5 M
0.5 M
20x SSC
Na3 citrate
NaCl
pH 7.0
0.3 M
3M
Primary wash buffer containing urea
Urea 360 g
6M
SDS 4 g
0.4%
20 x SSC 25 ml
0.5 x SSC
Make up to 1 litre. This can be kept for up to 3 months in a
refrigerator at 2–8°C. Stringency may be increased by using a lower
final SSC concentration, for example 0.1 x SSC instead of 0.5 x SSC.
Primary wash buffer without urea
SDS 4 g
0.4%
20 x SSC 25 ml
0.5 x SSC
Make up to 1 litre. This can be kept for up to 3 months in a
refrigerator at 2–8°C. Stringency may be increased by using a lower
final SSC concentration, for example 0.1 x SSC instead of 0.5 x SSC.
Secondary wash buffer (2x SSC)
20 x SSC
100 ml
Make up to 1 litre. This can be kept for up to 3 months at 2–8°C.
14
8. The ECL direct nucleic acid labelling
and detection system protocols
8.1. Protocol summary
1. Preparation of nucleic acid blot
2. Hybridization buffer preparation
Add salt, dissolve blocking agent (1.5–2 hours)
3. Pre-hybridization of blots
42°C for 15–60 minutes
4. Probe labelling
Denaturation for 10 minutes
Labelling reaction for 10 minutes
5. Hybridization
42°C for several hours (for example, colony screening) to overnight
(for example, single copy gene detection)
6. Blot washing
2 x 20 minutes for stringency washes, 2 x 5 minutes in SSC
7. Detection
Incubation in detection reagents (1 minute)
Exposure to Hyperfilm™ ECL (several minutes–2 hours)
15
8.2. Protocol A. Labelling, hybridization and
detection
Users are recommended to read the complete protocols before
starting work. The techniques of capillary or Southern blotting are
described in protocol B, page 30, Northern blotting in protocol C,
page 37 and colony/plaque blotting in protocol D, page 39. Before
commencing labelling, it may be found helpful to prepare the
hybridization buffer and begin pre-hybridizing the membrane (see
section 8.2.2.).
8.2.1. Preparation of labelled probe
The following protocol is for labelling 100 ng of probe DNA. However,
the amount of probe DNA required for a hybridization depends on
the size of blot to be probed, the amount of target DNA present and
the proportion of probe sequence within the DNA to be labelled.
In practice, blot size will determine the hybridization volume to be
used (see section 8.2.2.), while the amounts of target and probe
sequence will determine the probe concentration. 10 ng probe/ml
is appropriate for many applications, although this may be reduced
where there is a large amount of available target DNA, for example in
many plaque or colony screening experiments (protocol D).
The signal:noise ratio of the final result is sometimes lower when
whole clones are used as hybridization probes. Protocols for purifying
inserts from vectors can be found on pages 43–45. For labelling
probes in low melting point agarose see page 46. The method can be
used to label probes greater than 50 bp in length, although for single
copy gene detection, probes longer than 300 bp are recommended
to give the required sensitivity. The protocol is written for DNA probes,
but an identical procedure can be followed to label RNA probes.
16
Protocol
Notes
1. Dilute the DNA to be labelled
to a concentration of 10 ng/μl
using the water supplied.
1. The concentration of salt in
the sample of nucleic acid
should not exceed 10 mM.
The Hind III control DNA is
diluted ready for use (100 ng/10 μl).
2. Denature 100 ng of the DNA
sample (10 μl) by heating for
5 minutes in a boiling water
bath.
2. The minimum amount of DNA
which should be labelled is
100 ng in 10 μl. If more is
required the volume should
be increased appropriately
while maintaining the
concentration at 10 ng/μl. It is
essential to use a vigorously
boiling water bath rather than
a heating block for complete
denaturation.
3. Immediately cool the DNA on
ice for 5 minutes. Spin briefly
in a microcentrifuge to collect
the contents at the bottom of
the tube.
3. Maintaining the DNA in
a single stranded form is
required to ensure a good
labelling efficiency. In general
denaturation of RNA is also
advised to avoid secondary
structure.
4. Add an equivalent volume of
DNA labelling reagent (10 μl) to
the cooled DNA. Mix gently
but thoroughly.
4. The volume of labelling
reagent should be equal to
the volume of DNA to be
labelled. For example 100 ng
of DNA in 10 μl will require
10 μl of labelling reagent.
17
Notes
Protocol
5. Add the glutaraldehyde
5. For example, 10 μl DNA
solution, use a volume
solution (10 ng/μl) + 10 μl
equivalent to the volume of
labelling reagent will require
the labelling reagent (10 μl).
10 μl glutaraldehyde solution.
Mix thoroughly. Spin briefly in a
It is important to maintain the
microcentrifuge to collect the
ratio of DNA labelling reagent:
contents at the bottom of the
glutaraldehyde to ensure
tube.
a good labelling efficiency.
Alteration to this ratio can
result in high backgrounds and
poor sensitivity.
6. Incubate for 10 minutes at
37°C.
6. When labelling nucleic
acids <300 bp, increasing
the incubation period to
20 minutes may improve
labelling efficiency.
7. If not used immediately, the
probe can be held on ice for
a short period, for example
10–15 minutes.
7. Labelled probes may be stored
in 30% glycerol at -15°C to -30°C
(see page 43) for up to six
months. Further treatment of
the probe before use is not
required.
18
8.2.2. Hybridization and stringency washes
The hybridization and wash conditions given in the following protocol
are appropriate for a majority of probes, allowing detection of single
copy mammalian genes without significant cross-hybridization to
non-homologous sequences.
Protocol
Notes
1. Prepare the hybridization
buffer as follows:
At room temperature, take
the required volume of
hybridization buffer. Add solid
sodium chloride (analytical
grade) to a concentration
which is most suitable for
effective probe hybridization.
Add the blocking agent to
a final concentration of
5% (w/v). Immediately mix
thoroughly to get the blocking
agent into a free suspension.
Continue mixing at room
temperature for 1 hour on
a magnetic stirrer or roller
mixer, then heat to 42°C for
0.5–1 hour with occasional
mixing.
1. 0.25 ml/cm2 is recommended
for small blots and
0.125 ml/cm2 for large blots.
2. If necessary pre-heat
the required volume of
hybridization buffer to 42°C,
place the blots in the buffer
2. Hybridization can be carried
out in bags or boxes, provided
there is sufficient buffer for
the container. Adequate
The optimum sodium chloride
concentration may be
different for different probes.
0.5 M NaCl generally gives
acceptable results. If excess
buffer is prepared aliquots
should be pipetted into sterile
plastic containers and stored
at -15°C to -30°C. Prepared
buffer may be stored for at
least three months.
19
Protocol
Notes
2. Continued
and prehybridize for at least
15 minutes at 42°C with
gentle agitation. A 1 hour
prehybridization is routinely
used at GE Healthcare.
2. Continued
circulation of the buffer is
essential when hybridizing
several blots together and
it is important that the blots
are allowed to move freely.
High backgrounds can
result if there is insufficient
hybridization buffer or
movement of the blots.
When placing a blot on the
surface of the hybridization
buffer, allow it to saturate
completely. Avoid trapping
air between the blot and the
buffer, and finally submerge
the blot in the buffer. This is
particularly important when
hybridizing several blots
together.
3. Following pre-hybridization,
add the labelled probe to
the pre-hybridization buffer.
Avoid placing it directly on the
membrane and mix gently.
Some of the buffer can be
withdrawn for mixing with the
labelled probe before addition
to the buffer. Continue
incubation with gentle
agitation overnight at 42°C.
3. It is important not to exceed
42°C during hybridization;
higher temperatures are
deleterious to the enzyme
component of the DNA
labelling reagent. It is possible
to use shorter hybridization
times. Some loss of sensitivity
is likely with low target
applications, for example
20
Protocol
Notes
3. Continued.
single copy gene analysis but
for high target applications,
for example colony and
plaque blotting (protocol D),
shorter hybridizations of
1–4 hours are recommended.
4. Each primary wash should
not exceed 20 minutes. If
desired, primary wash buffer
without urea can be used.
This should be prewarmed to
55°C. 2 x 10 minute washes at
55°C are recommended. The
total wash time should not
exceed 20 minutes, to protect
peroxidase enzymatic activity.
Longer washing times at 55°C
can result in a significant
loss of sensitivity. Stringency
may be altered by the SSC
concentration in both types of
primary was buffers; 0.5 x SSC
is low stringency, 0.1 x SSC is
high stringency (see page 14).
Several blots can be washed
in the same solution provided
that they can move freely.
Stocks of the primary wash
buffer are stable at 2–8°C for
up to 3 months.
4. Prepare the primary wash
buffer (see page 14) and
preheat to 42°C. This is used
in excess at a volume of
approximately 2–5 ml/cm2 of
membrane. Carefully transfer
the blots to the solution and
wash for 20 minutes with
gentle agitation, ensuring
temperature does not exceed
42°C.
21
Protocol
Notes
5. Carry out a further wash in
fresh primary wash buffer at
42°C for 20 minutes.
6. If necessary blots may be
left in secondary wash buffer
for up to 30 minutes at room
temperature before detection.
Hybridized blots may be
stored overnight wetted
with secondary wash buffer
wrapped in SaranWrap™, in a
refrigerator at 2–8°C. Do not
allow the blot(s) to dry out.
6. Place the blot(s) in a clean
container and add an excess
of secondary wash buffer
(See page 14). Wash with
gentle agitation for 5 minutes
at room temperature. Carry
out a further wash in fresh
secondary wash buffer
at room temperature for
5 minutes.
22
8.2.3. Signal generation and detection
Please read through this whole section before proceeding. For
optimum sensitivity, it is necessary to work with reasonable speed
once the blots have been exposed to the detection solutions, as there
is no lag phase with the ECL reaction. All steps can be carried out in
a darkroom if desired; it is necessary to switch off the light only after
step 5).
Equipment which is needed includes an X-ray film cassette, a roll of
SaranWrap (other ‘cling-flims’ may be suitable), a timer and bluelight sensitive autoradiography film, for example Hyperfilm ECL
(RPN2103/2104). If possible wear powder free gloves. Powder from
gloves can inhibit the ECL reaction leading to blank patches on film.
The exposure times recommended below are appropriate for
Southern blot applications such as the detection of single copy
mammalian genes. Shorter exposures can be used for high target
applications such as colony or plaque lifts or the detection of
amplified target.
Protocol
Notes
1. Mix an equal volume of
detection reagent 1 with
detection reagent 2 to give
sufficient solution to cover
the blot. 0.125 ml/cm2 is
recommended.
1. Mix only the volume of
detection reagents necessary
to cover the blot. Storage
of the mixed solution is
not recommended. If not
used immediately, prepared
detection solution can be
held on ice for a short period,
for example 30 minutes.
Avoid cross-contamination of
reagents 1 and 2.
23
Protocol
Notes
2. Drain the excess secondary
wash buffer from the blots
and place them in fresh
containers or on a sheet
of SaranWrap, DNA side
uppermost. Add the detection
reagent directly to the blot(s)
on the side carrying the DNA;
do not leave the blots to dry
out.
2. An alternative method is to
equilibrate the drained blots
in a half quantity of reagent 2
(0.0625 ml/cm2), add to this a
similar quantity of reagent 1,
and mix by gentle shaking.
3. Incubate for 1 minute at room
temperature.
4. Drain off excess detection
reagents and wrap the blots
in SaranWrap. Gently smooth
out air pockets.
4. Excess detection reagent can
be removed by gently laying
the blots, DNA side up, on a
folded tissue or Whatman™
3 MM paper for a few seconds.
5. Place the blots DNA side up,
in the film cassette. Work
as quickly as possible to
minimize the delay between
incubating the blots in
substrate and exposing them
to the film (next step).
5. Ensure there is no free
detection reagent in the film
cassette; the film must not
get wet.
6. Switch off the lights and place on
a sheet of autoradiography
film (for example Hyperfilm
ECL) on top of the blots. Close
the cassette and expose for
30 seconds.
6. This should be carried out in
a dark room, using red safe
lights. Do not move the film
whilst it is being exposed.
Hyperfilm ECL can be preflashed if required (e.g. using
24
Protocol
Notes
6. Continued.
a Sensitize™ pre-flash unit,
RPN 2051). This can give
a significant improvement
in sensitivity at low levels.
The DNA side of the filter
(wrapped in SaranWrap) must
be placed next to the film for
maximum sensitivity.
7. Remove the film and develop.
If required, expose a second
film for an appropriate length
of time.
7. For initial experiments with
the ECL direct system, an
exposure of 30 minutes is
recommended with low
target applications. A further
exposure of 60–120 minutes
should give an equivalent
image. An alternative
approach is to carry out a
short 1–2 minute initial
exposure and use this
to judge the length of
subsequent exposure.
When undertaking colony
and plaque screening it
is important to use the
minimum film exposure
time which enables positive
colonies/plaques to be
unambiguously detected.
25
8.2.4. Hybridization and stringency washing in tubes
Nucleic acid hybridization and stringency washing may be
conveniently performed in an incubator with an integral rotisserie
device or in an hybridization oven. These ovens allow the continuous
movement of fluid over the hybridizations which can therefore
be performed in minimal volumes so that they are particularly
economical on probe usage. The procedure outlined below is
intended as a general guideleine for the use of hybridization ovens.
It may require optimization depending on the application and
equipment available.
Notes
Protocol
1. Preheat the hybridization
buffer (section 1 page 19) to
42°C.
2. In a suitable container prewet the blot in 5x SSC (75 mM
Na citrate, 0.75 M NaCl,
pH 7.0). Loosely roll the blot
and place inside the tube.
Add a small amount of 5x SSC
to the tube and unroll the
blot ensuring no air bubbles
are trapped between the
membrane and the tube. Do
not allow the blot to overlap
itself.
2. It is important to exclude any
bubbles trapped between the
hybridization tube and the
membrane as this can lead to
black patches on detection.
Nylon mesh may be used if
there is significant overlap of
the blot. Sandwich the blot
between two layers of nylon
mesh. Roll the sandwich
(ensuring no air bubbles are
trapped) with the nucleic acid
facing inwards. Place the roll
in the tube so that it ‘unrolls’
in the opposite direction from
26
Protocol
Notes
2. Continued.
the movement of the
rotisserie. Again ensure that
no air bubbles are trapped.
Nylon mesh may be reused
following washing in a 1%
SDS solution and thorough
rinsing in distilled water.
3. A hybridization volume of
0.0625–0.125 ml/cm2 is
recommended. In practice,
the tube size will determine
the minimum volume to
be used; for example the
minimum volume for use with
a 4 cm diameter and
30 cm long is 20 ml. Generally
more buffer is required than
would be used with standard
radioactive hybridizations.
High backgrounds can
result if there is insufficient
hybridization buffer.
3. Pour off the 5x SSC and add
the appropriate volume of
hybridization buffer.
4. Prehybridize in a rotisserie
oven for at least 15 minutes
at 42°C.
5. Prepare the labelled nucleic
acid probe as in section 1).
6. Some of the buffer can be
withdrawn for mixing with the
6. Add the labelled probe to the
prehybrization buffer, but
27
Protocol
Notes
6. Continued.
avoid placing it directly on
to the membrane. Hybridize
overnight in a rotisserie oven
at 42°C.
6. Continued.
labelled probe before addition
to the bulk of the buffer.
7. Prewarm the appropriate
volume of primary wash
buffer to 42°C.
7. If desired, primary wash
buffer without urea may
be used. This should be
preheated to 55°C.
8. Discard the hybridization
buffer. Add 50–100 ml 5x SSC
to the tube and replace in the
rotisserie oven for 5 minutes.
8. When washing with primary
wash buffer without urea,
use this period to increase
the temperature of the
hybridization oven to 55°C.
This should take 5–10 minutes
depending on the apparatus.
9. Discard the 5x SSC and
replace with primary
wash buffer so that the
hybridization tube is
approximately one-third
full. Return the tube to the
rotisserie oven and wash the
blot for 20 minutes at 42°C.
9. When using primary wash
buffer without urea, wash for
10 minutes, followed by
2 x 5 minutes at 55°C. The
total wash time should
not exceed 20 minutes.
Stringency may be altered
by adjusting the SSC
concentration in both types
of primary wash buffer (see
section 2).
10. Discard the primary wash
buffer and replace with an
equivalent volume of fresh
buffer. Was for 10 minutes
28
Protocol
Notes
10. Continued.
at 42°C. Repeat this wash a
second time.
11. Remove the blot from the
hybridization tube, place in a
suitable container and cover
with an excess of secondary
wash buffer. Incubate
with gentle agitation for a
further 5 minutes at room
temperature.
12. Discard the secondary wash
buffer and replace with an
equivalent volume of fresh
secondary wash buffer.
Incubate with agitation for
a further 5 minutes at room
temperature.
13. Carry out the detection as
outlined in section 8.2.3.
29
8.3. Protocol B. Southern blotting
8.3.1. Gel electrophoresis
Protocol
Notes
1. Make a 1% agarose solution
in 1x TAE buffer (see page 13).
1. Heating to approximately 90°C
is required to dissolve agarose.
Low electroendosmosis grade
agarose is recommended
(for example, Agarose NA,
17-0554-01, -02, -03).
Different concentrations of
agarose can be used, ranging
from 0.8%–2% depending on
the size of the fragments to
be separated.
2. Cool agarose solution to
50–60°C. Add ethidium
bromide, mix, pour into gel
former, and insert gel comb(s)
to produce wells of up to
5 mm in width, removing any
air bubbles.
2. Use 1 μl of a 10 mg/ml
solution of ethidium bromide
per 60 ml of agarose solution.
Care should be taken to avoid
skin contact with this reagent.
3. Allow to set and place gel
in electrophoresis tank. Fill
tank with gel running buffer,
sufficient to cover surface of
the gel.
3. Gel running buffer: 1x TAE
as for agarose. Gel running
buffer must be fresh, do not
reuse. The gel running tank
must be clean. Failure to
ensure cleanliness of the tank
and buffer may result in the
appearance of a black band at
the top of the blot on detection.
30
Protocol
Notes
4. Apply DNA samples to wells in
gel. The diluted λ DNA should
be heated for 5 minutes at
65°C and then snap-cooled
on ice before loading. This
dissociates the cos ends.
4. Loadings of 2–10 μg of
restriction enzyme digested
genomic DNA are usually
sufficient. The control DNA (λ
DNA Hind III digest) supplied
in the kit (10 μg/ml) should be
loaded at 100 pg per well. 1x
TAE buffer is a suitable diluent
for the DNA and the inclusion
of high density mixture (at
20% of the final volume)
containing xylene cyanol and
bromophenol blue dye (see
page 13) aids loading the
samples into the gel. Sheared
herring sperm DNA (2.5 μg)
may be added to the diluted
(100 pg) control DNA as a
‘carrier’, but it is not essential.
5. Apply current to gel and allow
to run until bromophenol blue
indicates that sample has
run for a sufficient distance,
for example two thirds of the
way down the gel.
5. A voltage of, for example,
60 volts over 4–5 hours for a
20 cm long gel can be used
for genomic DNA. Overnight
electrophoresis runs can be
achieved by using 1 V/cm
of gel.
31
8.3.2. Processing the gel
Protocol
Notes
1. Slide gel from gel-former into
a suitable plastic box or a
glass/pyrex dish.
1. With UV illumination,
photograph the gel to record
the electrophoretic separation
of the loaded DNA samples/
controls.
2. Cover with depurination
solution (see page 13) and
begin agitation, for example
using an orbital shaker,
ensuring that the gel moves
freely. Treatment should be
stopped immediately the
bromophenol blue-dye has
turned completely yellow
(10–12 minutes).
2. Approximately 400 ml is
recommended for a 20 cm
x 20 cm gel. Depurination
is not needed if only small
fragments (<10 kb) are to be
transferred.
3. Discard depurination solution
and rinse gel with distilled
water.
4. Cover gel with denaturation
solution (see page 14) and
begin agitation. Agitate
for 25 minutes after the
bromophenol dye has
returned to its blue colour.
4. Use a similar volume to that
used for depurination.
5. Discard solution and rinse gel
with distilled water.
32
Protocol
Notes
6a. Hybond™ N+: Cover gel
with neutralizing solution
(see page 14) and begin
agitation. Continue agitation
for 30 minutes.
6. Use a similar volume to that
used for depurination and for
denaturation. While the gel is
neutralizing begin to prepare
for the capillary blotting.
6b. Hybond ECL (nitrocellulose):
Cover gel with neutralizing
solution (see page 14) and
begin agitation. Continue
agitation for 30 minutes.
Discard solution and replace
with fresh neutralizing
solution. Continue agitation
for a further 15 minutes.
8.3.3. Capillary blotting
Protocol
Notes
1. Part fill a glass/pyrex dish
with 20x SSC, and make
a supporting platform (for
example using a glass plate
or a perspex gel former).
Cover it with a wick made
from three sheets of filter
paper (for example Whatman
3 MM saturated with 20x SSC).
1. There is a diagram of the
capillary blotting stack on
page 34. Overlay the filter
paper with cling film to
enable air bubbles to be
smoothed out. A glass rod
or a pipette can be used
for this and for subsequent
similar operations. the cling
film should be removed
afterwards.
2. Place the gel on the 3 MM
paper, taking care to avoid
trapping air bubbles.
33
Protocol
Notes
2. Continued.
Surround with cling film
to prevent the SSC being
absorbed directly by the
paper towels.
3a. Hybond-N+: Cut a sheet
of Hybond-N+ nylon
membrane to the size of
the gel.
3. Care should be taken to avoid
contaminating the membrane
by touching it. Gloves should
be worn.
3b. Hybond ECL: Cut a sheet of
Hybond ECL nitrocellulose
membrane to the size of the
gel. Pre-wet the membrane
by briefly immersing in
distilled water, followed by
soaking in 20x SSC for at
least 10 minutes.
4. Place the membrane on top
of the gel, without trapping
air bubbles.
4. The membrane should not
come into direct contact with
the 3 MM paper wick.
5. Place three sheets of 3 MM
paper, cut to size and wetted
with 10x SSC on top of the
membrane, again avoiding
trapping air bubbles.
6. Place a 5–7 cm stack of
absorbent paper towels on
top of the 3 MM paper.
34
Notes
Protocol
7. Place a glass plate on top
of the paper towels and
apply a weight (for example
approximately 750 g for a
20 cm x 20 cm gel). Leave
overnight (or for at least
4 hours).
8.3.4. Processing the blot
Protocol
Notes
1. Dismantle blotting stack
down to membrane, remove
membrane and gel together,
place membrane side down
on a clean piece of 3 MM
paper. Peel off gel and
discard. Mark the membrane
so that the face carrying the
DNA is distinguishable.
1. A soft-lead graphite or a
chinagraph pencil can be
used to mark the membrane.
2a. Hybond-N+: Place
membrane in 6x SSC and
rinse for 1 minute with
gentle agitation to remove
agarose. There are several
alternative methods
which may be used for
DNA fixation, but for ECL
detection either baking or UV
treatment is recommended.
i) Baking (80°C for 2 hours)
2a. Hybond-N+: If not to be used
immediately, fixed blots can
be stored dry wrapped in
SaranWrap at 2–8°C, or, for
up to several weeks at room
temperature under vacuum.
35
Protocol
Notes
2a. Continued.
as described for Hybond
ECL below. A vacuum is
not required with nylon
membranes.
ii) UV fixation optimize for
each transilluminator/cross
linker.
For RNA blots baking is
the recommended fixation
method as described in i)
above. UV fixation can also
be used.
2b. Hybond ECL: Do not exceed
a temperature of 80°C.
While blot is baking prepare
to label probe and set up
hybridization (section 8.2.). If
not to be used immediately,
baked blots can be stored
dry at either 2–8°C or at
room temperature under
vacuum.
2b. Hybond ECL: Lay filter, DNA
side up, on a clean piece
of 3 MM paper and allow
surface liquid to drain, then
remove to a second piece
of 3 MM paper and allow
to air dry. Sandwich the
blot between fresh 3 MM
paper and bake dry (80°C for
2 hours) in a vacuum oven.
8.3.5. Probe labelling, hybridization and detection
Notes
Protocol
1. Probe concentrations, buffer
volumes and detection
times given in section 8.2.)
are appropriate for both
Southern and Northern blots.
1. follow protocol A, sections
8.2.1.–8.2.3. inclusive.
36
8.4. Protocol C. Northern blotting
Electrophorese RNA in a denaturating system. Formaldehyde/agarose
is recommended for use with Hybond membrane
8.4.1. Gel electrophoresis
Protocol
Notes
1. Prepare gel; for example to
prepare 300 ml of gel add:
Agarose
3–4.5 g
10x MOPS buffer 30 ml
Water
219 ml
1. 250 ml is sufficient for
4000 cm2 gel. Thin gels are
more desirable than thick
gels.
Dissolve agarose by heating
in a microwave or on a
steam bath, mix well and
cool to 50°C. In a fume hood,
add 16.2 ml of 37% (v/v)
formaldehyde, mix well, add
20 μl of a 10 mg/ml solution
of ethidium bromide, mix by
swirling. Working in a fume
hood, pour the gel into the gel
former, and allow to set.
2. Prepare RNA samples:
Loadings are normally
10–20 μg of total RNA, or less
for an abundant mRNA. 1–2 μg
or less is usually sufficient for
poly(A)+ RNA. The RNA in 5 μl
or less of buffer is diluted with
15 μl of RNA loading buffer
37
Protocol
Notes
2. Continued.
(see page 13). Boil sample,
in a vigorously boiling water
bath, for 2 minutes. Chill
samples on ice and then load
on to gel.
3. Running the gel: Running
buffer is 1x MOPS buffer.
Run gel at 5–7.5 V/cm
for 2–3 hours, or until the
bromophenol blue dye has
migrated three-quarters of
the way down the gel. The gel
may now be photographed
under UV illumination.
8.4.2. Treating the gel
Rinse the gel for 3 x 10 minutes in 10x SSC to remove formaldehyde
from the gel. Use a volume sufficient to float the gel in the container.
8.4.3. Blotting the gel
Follow the procedure in Southern blotting protocol, section 8.3.3.,
‘Capillary blotting’, and section 8.3.4., ‘Processing the blot’, taking care
to read the notes for Northern blotting.
8.4.4. Probe labelling, hybridization and detection
Protocol
Notes
1. Follow protocol A sections
8.2.1.–8.2.3. inclusive.
1. Probe concentrations, buffer
volumes and detection
times given in protocol A are
appropriate for both Southern
and Northern blots.
38
8.5. Protocol D. Colony and plaque blotting
The following protocols describe the application of the ECL direct
system to colony and plaque screening. The recommended probe
concentrations, hybridization times and film exposure times, whilst
generally applicable, are intended as guidelines; some optimization
may be necessary for individual systems.
8.5.1. Plating out and incubation
Protocols
Notes
1. For chloramphenicol
amplifiable plasmids it
is possible to limit this
incubation to 5–6 hours,
followed by an overnight
amplification on
chloramphenicol plates.
1. Plate out cells or
bacteriophage in the normal
way and incubate overnight
at the required temperature.
8.5.2. Colony/plaque lifts
Protocols
Notes
1. Remove the agar plates
from the incubator. Label a
Hybond-N+ disc (RPN 82B) with
a chinagraph pencil, or other
suitable indelible marker.
Place the disc (labelled side
down) on the surface of the
plate, in contact with the
colonies, until the disc is
completely wetted.
1. For plaques (M13/λ) and
colonies (which are to be
chloramphenicol amplified),
leave the discs on the agar
plates for 1–5 minutes.
2. Mark the membrane and agar
using a sterile needle or other
2. For plaques (M13/λ) and
colonies (which are to be
39
Protocols
Notes
2. Continued.
means to ensure correct
orientation of colonies/
plaques.
2. Continued.
chloramphenicol amplified),
leave the discs on the agar
plates for 1–5 minutes.
3. Remove the disc from the
agar plate. If no amplification
is to be performed, proceed
directly to section 8.5.3. step 1.
4. For colonies which are to be
chloramphenicol amplified,
the disc should now be
placed colony side up on an
L-agar/chloramphenicol
plate, and incubated at 37°C
overnight.
5. Remove the disc from the
agar plate.
8.5.3. Lysis and fixation
Protocols
Notes
1. Periods of longer than
1 minute for colonies will
increase the intensity of the
signal on detection, but may
allow the colonies to diffuse
slightly. There is no need for
this period to be longer than
5 minutes because of the high
amounts of target DNA.
1. Place the disc, colony/plaque
side up, for 5 minutes on
to two sheets of Whatman
3 MM paper which have been
saturated with 0.5 M NaOH.
40
8.5.4. Rinsing
Protocols
Notes
1. Place the disc into a dish
containing 400 ml 5x SSC
and agitate on an orbital
shaker for 1 minute. However,
discs of overnight growth
colonies which have not been
choramphenicol amplified,
require more vigorous
agitation/rinsing to remove
unwanted colony naterial.
It is important to remove
cellular debris at this stage to
avoid non-specific binding of
HRP-labelled probes during
hybridization. This may be
done by holding the disc
with forceps and agitating
vigorously by hand in the
buffer. Discs should be rinsed
twice in this manner.
1. This volume is sufficient for
rinsing up to 10 discs, for
amplified colonies use fresh
5x SSC every 10 discs. Due to
the large amount of bacterial
debris that is removed from
the discs of overnight growth
colonies, 400 ml of 5x SSC is
sufficient for only two discs.
2. Place the disc, DNA side up,
on 3 MM paper to dry.
3. The disc may now be used
in a hybridization or stored
under vacuum at room
temperature.
41
8.5.5. Hybridization and detection
A range of conditions have been used;
Plate concentration:
2–10 ng/ml
Length of hybridization:
1–4 hours
Film exposure times:
1 minute–1 hour
The following screening applications have been used:
Colonies: overnight growth (plasmids)
chloramphenicol amplification
(plasmids)
overnight growth (cosmids)
Plaques: M13 and λ
When performing a new screening experiment, it is suggested
that a 4 hour hybridization with 10 ng/ml of probe should be
used as a starting point. Reducing the hybridization time or probe
concentration will reduce sensitivity, but this can be tolerated in
most high copy number plasmid or M13 systems, because of the
high amount of target DNA. However, there is less target DNA in
cosmid or λ systems, so any reduction in hybridization time or of
probe concentration may result in the loss of signal strength from the
positive hybrids. Any reduction in signal strength can be offset with
longer film exposures. For probe labelling, hybridization and detection
follow the protocols in section 8.2., 8.2.1.–8.2.3. inclusive.
42
9. Additional Information
9.1. Quality control
Each batch of the system is checked by our quality control group to
ensure that it will detect 0.5 pg of target DNA in a genomic Southern
blot.
The performance of the system may be verified using the control
DNA provided.
9.2. Reprobing blots
Blots that have been used to generate a signal on film can be
reprobed several times, using different (or the same) DNA probes,
labelled in the same way as the original probe. Because the system
utilizes an enzyme label, and this becomes inactivated following the
chemiluminescent reaction, it is not necessary to ‘strip’ the blots of
old probe before starting second, and subsequent hybridizations.
The blot should be kept in the detection reagent for an extended
period (for example, overnight) to allow the lumiscence to decrease
to negligible levels before reprobing. The reprobing procedure begins
at section 8.2.1. of this protocol. Membranes should be kept moist
between reprobings, for example wrapped in SaranWrap, and stored
at room temperature.
The limit to the number of reprobings is likely to be governed by
physical damage to the blots. It is therefore recommended that blots
are always handled carefully.
The membrane must be blocked by using hybridization buffer
containing 5% (w/v) blocking agent during each reprobing.
9.3. Storage of labelled probes
In our own laboratories a number of DNA probes have been labelled
then stored in 50% glycerol at -15°C to -30°C for up to 6 months,
43
and then successfully used in hybridization on Southern blots. Whilst
labelled probes appear to be reasonably stable, the working life of
different probe preparations generated by the user may vary.
9.4. Excising inserts from vectors
After insert purification it is important to determine the DNA
concentration to ensure efficient labelling (section 8.2.1.). This can
be determined spectrophotometrically. Alternatively an aliquot can
be electrophoresed in an agarose gel alongside a sample of DNA of
known concentration; ethidium bromide staining with UV illumination
will allow the DNA concentration to be estimated.
a) Using DEAE-cellulose paper
Vectors containing inserts should be digested with appropriate
restriction enzymes and the DNA fragments separated in a 1%
agarose gel containing ethidium bromide. Examine the gel over UV
light and make horizontal slits above and below the band(s) to be
recovered. Insert prepared pieces of DEAE-cellulose paper (detailed
overleaf) into each slit to the full depth of the gel and squeeze
the gel firmly against the paper to close the incisions. The paper
above the band protects against subsequent contamination by
DNA fragments of higher molecular weight. Resume electrophoresis
until the DNA has entered the DEAE-cellulose paper strips, as verified
by examination over UV illumination. Remove the paper, cut off the
perimeter which does not contain DNA and wash in cold distilled
water for 5–10 minutes. Drain and blot dry with tissues. Place in a
1.5 ml polypropylene microcentrifuge tube, and add 300–700 μl of
extraction buffer (see below). Shred the paper by mixing the tube and
contents on a vortex mixer.
Incubate the tube at 37°C for 2 hours, with occasional agitation.
If required the tube may be stored at 2–8°C for up to 24 hours
prior to this incubation. Transfer the mixture to a capless 0.5 ml
polypropylene microcentrifuge tube which has a hole in the bottom,
44
made by piercing with a fine needle. Place the tube in a 1.5 ml tube
and centrifuge for 5 minutes. Centrifuge the filtrate for a further
3 minutes to pellet any remaining fibres. Extract the supernatant,
containing the DNA, with 3 volumes of butan-1-ol (saturated with
water) to remove ethidium bromide. Precipitate the DNA with
2 volumes of ethanol (from 1 hour to overnight at -15°C to -30°C,
centrifuge down the pellet, rinse in 70% ethanol (-15°C to -30°C) and
redissolve in distilled water. Up to 80% recovery can be achieved
when extracting small fragments of 2 kb and less.
DEAE-cellulose paper preparation
Cut strips of DEAE-cellulose paper (for example Whatman-DE81) to
a length equal to the gel thickness and slighly greater than the slot
width. Soak for sveral hours in 2.5 M NaCl. Wash the strips several
times in distilled water and store in 1 mM EDTA (pH8.0) at 2–8°C.
Extraction buffer (pH8.0)
1.5 M NaCl
1 mM EDTA
20 mM Tris HCl
b) Using electroelution
As in the above procedure, vectors containing inserts should be
digested with appropriate enzymes to release the inserts. The entire
digest can be loaded into a preparative well in a 1% agarose gel
containing ethidium bromide. The gel should be run until the insert is
clearly separated from the vector, as observed using UV illumination.
The insert band should be cut out with a scalpel and transferred to a
dialysis bag filled with TAE buffer. As much buffer as possible should
then be expelled from the dialysis bag, it should be sealed, and then
immersed in an electrophoresis tank, parallel to the electrodes, and
current should be applied (100 volts, 2 hours).
During this time DNA is electroeluted out of the gel and on to the
inner wall of the dialysis bag. It is released from the wall of the bag
45
by reversing the polarity of the current for 2 minutes. The eluted DNA
can then be aspirated from the dialysis bag using a Pasteur pipette,
and precipitated by adding 2 volumes of ice cold ethanol and leaving
to stand at -15°C to -30°C (1 hour to overnight). After centrifugation
the pellet should be rinsed with 70% ethanol (-15°C to -30°C) and
redissolved in distilled water.
9.5. Labelling probes in low melting point
agarose
Whilst highly purified probes are recommended for use with ECL
direct system, it is possible to label probes that have been separated
in low melting point agarose, without extracting the nucleic acid from
the agarose. Using gels up to 0.7% agarose enables insert bands
to be excised and the nucleic acid labelled directly by following the
recommended protocol (section 8.2.), but omitting the rapid cooling,
section 8.2.1., step 3.
The boiling step both melts the agarose and denatures the DNA. The
solution can be left briefly to cool at room temperature, and cold DNA
labelling reagent is then added to it. The agarose is now sufficiently
dilute not to re-solidify.
The glutaraldehyde cross-linking and 10 minute incubation at 37°C
should be carried out as normal. The labelled probe can then be used
in hybridizations.
46
10. Troubleshooting
Problem: No bands visible
Possible cause
Remedy
1. Possible failure in
transfer of target
DNA from gel to
membrane
1.1. Stain gel with ethidium bromide to
check for any remaining DNA
1.2. Check that protocol (section B) has been
followed closely
1.3. Include control λ DNA; for 100 pg loading
the fourth band down represents 4.4 kb
(9 pg) and the doublet 2.3 and 2.0 kb
(4.8 and 4.2 pg respectively).
2. Hybridization and
detection not
successful
2.1. Check hybridization conditions by
including suitable controls, for example
control λ DNA provided.
2.2. Check that probe labelling was
successful and that the labelling
reagents were used as directed, and
in the correct sequence. Check also
that the DNA was essentially salt free.
Labelling of DNA can be detected by its
altered electrophoretic mobility on an
agarose gel. Ensure that hybridization
temperature did not exceed 42°C.
2.3. Check that the detection reagents
are working; pre-mix small quantities
of detection reagent 1 and detection
reagent 2 (1 ml of each) and in the
dark add 1 μl of DNA labelling reagent.
Visible blue light should be produced.
47
Problem: Only faint bands visible
Possible cause
Remedy
1. Low level of signal
1. Pre-flashing the film will increase its
sensitivity to the signal and linearize its
response. This does however require care
as increased backgrounds may result.
Pre-flashing involves hypersensitizing
the film just before use by pre-exposure
to a short flash of light (approximately
1 msec). Conventional electronic
photographic flash units are suitable,
when attenuated with a diffuser and
Wratten 6B filter, to give a flash of the
required intensity to increase the 540
nm absorbance of the developed film
to 0.15 above that of the unexposed
film. Alternatively, an optimized preflash
system such as Sensitize™, RPN2051, can
be used.
2. Check the the hybridization temperature
did not exceed 42°C.
3. Expose the film for an extended period
(several hours).
4. Poor DNA transfer on to membranes; use
control λ DNA as above.
48
Problem: High background
Possible cause
Remedy
1. Non specific light
generation
1.1. Check that an error has not been made
with the quantities of DNA and labelling
reagent used in labelling.
1.2. The presence of labelled vector
sequences in the hybridization may
increase ‘noise’. Use purified insert as
probe whenever possible.
1.3. Ensure that the blot is evenly covered
with hybridization buffer during the
hybridization, and that there are no
trapped air bubbles.
1.4. Ensure that the labelled probe is not
added directly to the membrane.
Instead remove an aliquot of buffer
from the prehybridization and mix the
probe with it, before returning it to the
bulk of the buffer.
1.5. Ensure that all washing steps have been
carried out for the specified times at the
specified temperatures.
1.6. Expose the film for a minimum period
(an initial 1 minute period may be all
that is required)
1.7. Use high quality membranes:
Hybond ECL (RPN2020D or RPN82D)
is the recommended nitrocellulose.
Hybond-N+ is the recommended nylon
membrane.
49
Problem: High background continued.
Remedy
Possible cause
1. Non specific
light generation
continued.
1.8. Damage to the membrane can cause
non-specific binding of the probe.
Handle blots carefully with gloved
hands and blunt non-serrated forceps.
1.9. Use clean forceps to handle blot
after washing. Contamination with
hybridization buffer or primary wash
buffer prior to detection will elevate
background.
2. Poor blocking of
membrane
2.1. Ensure that the blocking reagent is fully
dissolved at 5% (w/v). Vigorous stirring
may be necessary.
2.2. When reprobing blots, the membrane
must be reblocked each time by using
hybridization buffer containing blocking
agent, from section 6 onwards.
Problem: Blank patches
Possible causes
Remedy
1. Contamination
from powder in
gloves
1. Wear powder-free gloves.
50
11. References
1. Whitehead, T. P., et al., Nature., 305, pp.158-159, (1983).
2. Renz, M. and Kurz, C., Nucl. Acids Res., 12, pp.3435-3444, (1984).
3. Heslop-Harrison, J. S., et. al., Heredity., 65, pp.385-392, (1990).
4. Bellanné-Chantelot, C. et al., Cell., 70, pp.1059-1068, (1992).
5. Einspanier, R. et. al., J. Reprod. Fert., 90, pp.439-445, (1990).
6. Abe, A. et al., J. Clinical Micro., 28, pp.2616-2620, (1990).
51
12. Related products
Hyperfilm ECL
Pack of 25 films, 18 cm x 24 cm
Pack of 25 films, 30 cm x 40 cm
Pack of 25 films, 10 inches x 12 inches
Pack of 25 films, 5 inches x 7 inches
RPN2103
RPN2104
RPN1681
RPN1674
Hybond-N+ and Hybond ECL
Part of an extensive range of membranes available from GE Healthcare
ECL random prime labelling and detection systems
Based on the incorporation of a hapten (fluorescein) with detection by ECL:
15 labelling reactions; ECL detection reagents for
1000 cm2 membrane
RPN3029
30 labelling reactions; ECL detection reagents for
2000 cm2 membrane
RPN3030
60 labelling reactions; ECL detection reagents for
4000 cm2 membrane
RPN3031
ECL 3’-oligolabelling and detection system
For tailing oligos with fluorecein-dUTP.
Sufficient for 500 pmols oligonucleotides
ECL detection reagents for 4000 cm2 membrane
RPN2131
ECL 5’-thiol oligolabelling and detection system
For the direct labelling of thiol modified oligonucleotides
with horseradish peroxidase with detection by ECL
Sufficient for 5 x 5 μg oligonucleotide ECL detection
RPN2113
reagents for 4000 cm2 membrane
C6-thiol modifier
RPN2112
ECL probe-amp reagents
For non-radioactive DNA probe labelling during PCR*
6 reactions
RPN3020
12 reactions
RPN3021
52
53
54
55
GE Healthcare
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Uppsala
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Fax: +43 1 97272 2750
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Sanken Bldg. 3-25-1
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http://www.gehealthcare.com/lifesciences
GE Healthcare UK Limited
Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA
UK
imagination at work
RPN3000PL Rev E 2006